Extracellular vesicles (EVs) are small, membrane bound vesicles that transport cargoes such as proteins, RNA, DNA, and lipids between cells. EVs are universally released from tissues within the body, and trafficking of EVs within the nervous system is important for cell-cell communication. EVs can also contribute to the disposal or spread of pathological proteins in neurodegenerative disease, making understanding the biology of EV regulation within the nervous system highly relevant to human health. Our knowledge of EV biology is primarily based on studies carried out using in vitro or cell culture-based systems. While these studies have identified a number of important molecular players that are likely involved in these processes, the molecular mechanisms driving EV cargo uptake by target cells, in a complex, in vivo nervous system remains unstudied. The Drosophila NMJ was the first system to study in vivo trafficking of EV cargoes, and our lab has developed novel tools to track endogenously labeled, neuronally derived EV cargoes as they get transferred to and taken up by the postsynaptic muscle cell. This level of detail within an in vivo complex nervous system cannot be attained in another system, and when combined with its genetic tractability, amenability to imaging, and available cell-biological, and tissue specific manipulations, the Drosophila NMJ is the ideal model system with which to ask direct questions about EV cargo uptake mechanisms. Using this system, we have identified a role for the Drosophila membrane-remodeling protein Amphiphysin in the regulation of postsynaptic EV cargo distribution. The goal of this proposal is to determine how Amph contributes to EV cargo uptake in vivo, and to define the cell-biological pathways that regulate EV cargo uptake in vivo in a complex nervous system. This is particularly intriguing because in humans, mutations in Amphiphysin-2/BIN1 are a very strong genetic risk factor for late onset Alzheimer's disease, and disruption of EV cargo trafficking is implicated in neurodegenerative disease. Together, this makes defining the mechanisms of Amph in EV cargo uptake, and how these are regulated at the cell-biological level, critical for understanding how Amph, and EV cargo uptake may contribute to neurological disease processes. Carrying out the experiments outlined in this proposal will provide me with training in high resolution microscopy, quantitative image analysis, cell- biological manipulations, and advanced Drosophila genetics. Furthermore, it includes concrete plans to enhance my training in leadership, mentorship, the responsible conduct of research, and scientific communication. Together, training in these areas will prepare me to become an independent scientist.